There are a number of known specific binding assay methods such as an immunoassay in which an antigen-antibody reaction is employed, a receptor assay in which a receptor is used and a nucleic acid probe assay in which hybridization of complementary nucleic acid sequences is employed. Because of the high specificity, these assay methods are used frequently in various fields including clinical inspection and the like.
In general, these methods are divided into a heterogeneous method which requires a step for the isolation of unreacted substances after specific binding reactions and a homogeneous method that does not require such an isolation step.
The heterogeneous method is useful for the measurement of samples with relatively high sensitivity and can be regarded as a general purpose method, because it can detect the degree of the specific binding reaction without the interference of unreacted substances. This method, however, requires complex handling for the removal of unreacted substances and, in some cases, requires special tools or instruments such as a washing apparatus. Because of this, it is still necessary to improve this method in terms of its simplicity and rapidity.
With the aim of improving the handling simplicity of the homogeneous type assay method, various techniques have been developed, which include agglutination method, EMIT method, proximal linkage immunoassay such as an enzyme channeling technique, immunochromatographic assay and the like. These techniques, however, are still inferior to the heterogeneous method in terms of efficiency and general purpose use.
For example, the aggtutination type methods which are effected by the formation of aggregates by antigen-antibody reaction or by the inhibition of the aggregate formation, such as a gel diffusion technique, an erythrocyte agglutination technique, a latex agglutination technique and the like, have considerably inferior sensitivity and accuracy in comparison with other methods in which labels such as enzymes are used.
Examples of homogeneous type methods which are effected by the use of labels such as enzymes include: EMIT method and the like which are effected by the modulation of enzyme activity, itself based on a specific binding reaction; and proximal linkage immunoassay in which signal modulation occurs when two types of labels having a mutual relation get close due to a specific binding reaction. In the case of the EMIT method and related techniques, it is necessary to modify an enzyme, a coenzyme component, an enzyme inhibitor or the like with a substance to be assayed (or an analogue of the substance to be assayed) while the activity of the enzyme or the like is kept constant and to induce a significant modulation of the enzyme activity through a binding reaction of the modified product with a specific binding substance. Because of such requirements, this method is inferior to other methods in terms of sensitivity and general purpose use and, in some cases, cannot be used depending on the physical properties (molecular weight, configuration, solubility and the like) and chemical properties (reactivity, position of functional groups and the like) of the substance to be assayed. On the contrary, the proximal linkage immunoassay can sometimes be effected by certain labeling techniques which are generally used in the heterogeneous method and therefore is superior to the EMIT method and the like in terms of general purpose use. In addition, from the viewpoint of specific binding reaction and detection of signals, increase in the sensitivity and decrease in the measuring time can be expected as the concentration of specific binding substances including labeling substances increases. Such a concentration increment, however, increases approaching frequency of labels independent of the specific binding reaction. Such properties which are contrary to each other become a significant factor that limits the sensitivity. An example of the proximal linkage immunoassay is the enzyme channeling technique in which two types of enzymes are used that catalyze two continued reactions such as formation of a product by a first enzyme and utilization of the product by a second enzyme as its substrate. Processes in relation to such a technique have been disclosed for instance in GB 2018986, EP 32286, EP 75379, Analytical Biochemistry (vol. 106, pp. 223-229, 1980) and the like. However, each of these prior art processes is based on the modulation of the total enzyme reaction rate, which is effected by a difference between a time when a labeled enzyme exists in a free state and a time when a microscopic environment is formed by a proximity effect of labels by a specific binding reaction so that channeling of the labeled enzymes can be carried out, or when the labeled enzyme undergoes specific binding to a dispersion type discrete particulate carrier (dextran particles or the like) or to a non-dispersion type carrier (filter paper or the like) which provides a channeling environment. In consequence, a free labeled enzyme in the liquid phase could approach the solid phase by free diffusion, and its non-specific reaction would cause a problem when the concentration of the labeled enzyme is high. Another problem of this technique is that, since a first enzyme can continue its reaction independent of the degree of proximity of a second enzyme, the reaction product of the first enzyme accumulates inside the measuring system when an environment for its channeling with the second enzyme is not formed. Such an accumulation of the product formed by the reaction of the first enzyme also causes acceleration of non-specific reactions. In order to solve such problems, a scavenger is used which can remove excess amounts of the first reaction product. However, addition of a scavenger in an effective amount causes a certain competitive inhibition effect on the whole reaction. Application of such a scavenger, therefore, cannot be regarded as a general purpose countermeasure.
A different type of assay method has been reported which includes a step in which a test sample presumably containing a substance to be assayed is permeated and developed, together with or separately from a labeled specific binding substance, in a chromatographic area that comprises a porous or particulate-packed type carrier to which a specific binding substance is immobilized. Such a method, or an immunochromatographic assay, is advantageous in some cases from the viewpoint of sensitivity and measuring time because of the large immobilizable effective surface area and the relatively high collision efficiency between reaction molecules which can induce a specific binding reaction, in comparison with a liquid phase reaction. In this instance, the label itself may be a signal substance or may generate a signal, moreover, instead of the generation of a signal by the label itself, a signal substance which is concerned in the generation of a signal may be generated by another label. Examples of the former type of labels include a radioactive isotope, a coloring substance, a fluorescent substance, a luminescent substance and the like, and those of the latter type of labels include an enzyme and the like. Processes for the immunochromatographic assay in which the former type labels are used have been disclosed for instance in DE 2729872, WO 8808534, EP 323605, and those in which the latter type labels are used have been disclosed for instance in EP 100619, WO 8505451 and U.S. Pat. No. 4,956,275. The latter type of labels may be advantageous in general when it is desirable to maintain sensitivity without using a radioactive isotope. In the case of the use of the latter type labels, a signal substance is generated by the contact of a component which is necessary for the signal substance generation (for example, an enzyme substrate, a coloring matter or the like) with a label (an enzyme or the like Because of this, such a process requires an additional step to effect the just described contact reaction (to be referred to as "development step" hereinafter), which is carried out after the permeation development step of a test sample presumably containing a substance to be assayed and a labeled specific binding substance, thus entailing the problem of complex handling.
In addition, these immunochromatographic assay processes have a common problem which still remains unsolved. That is, when a labeled specific binding substance and a liquid test sample are subjected to a chromatographic specific binding reaction in a specific binding reaction area, the specific binding reaction at least depends on the reaction frequency of two molecules. Because of this, the binding distribution of the labeled specific binding substance in the chromatographic direction in the specific binding reaction area becomes, more or less, a gentle curved pattern such as a gradient pattern or a sigmoid pattern. When the concentration of a substance to be assayed in a liquid test sample is changed, the reaction frequency changes and the binding distribution of a label in the specific binding reaction area changes, but still exhibits a curved distribution pattern as a whole. Because of this, a boundary line between colored and un-colored portions becomes unclear in many cases, thus causing considerable errors when, for example, the concentration of a substance to be assayed in a liquid test sample is determined qualitatively or quantitatively based on the length or position of a colored portion after the development step, that is, the distance or position to the end point of the binding distribution of a label. In order to improve measuring accuracy, it is necessary to perform quantification of the binding distribution of the label in the specific binding reaction area by understanding it collectively. U.S. Pat. No. 4,956,275 discloses arrangement of a plurality of detection means in a detection area which is also used as a specific binding reaction area. By such an arrangement of a plurality of detection means in the detection area, understanding the accuracy of the binding distribution of a label in the specific binding reaction area is improved in comparison with the case of the use of a single detection means. However, such an improvement is not substantial, because the assay apparatus becomes complex and the number of detection means to be arranged is limited. The just cited patent also discloses that an enzyme channeling-based signal generation system can be arranged in the detection area. According to the description, however, the detection area means an area where immobilized antibody molecules are located, and the description merely explains that only an existing portion of a labeled second enzyme can generate a signal by immobilizing a first enzyme together with the antibody to the detection area and by adding a substrate of the first enzyme to a development solution. In consequence, the process of the just cited patent can simplify the development step, but cannot solve the aforementioned problems in terms of the measuring accuracy and sensitivity.
A specific binding assay device which comprises multilayer analytical materials is known as another type of the immunochromatographic assay. Techniques in relation to this type of process have been disclosed for instance in EP 51183, EP 66648, DE 3227474 and EP 236768. Of these, each of EP 51181 and EP 66648 discloses a process in which a specific binding layer comprising a porous material immobilizing a specific binding substance is superposed on a detection layer and, after completion of the specific binding reaction, a labeled substance permeated into the detection layer is detected with a reagent contained in the detection layer. These patents also disclose that a light-shading layer is interposed between the detection layer and the specific binding layer, in order to avoid a bad influence of signals originating from a labeled substance bound to the specific binding layer. However, when a signal product is formed by the reaction of a labeled substance with a reagent in the detection layer, such as the case in which the label is an enzyme, the light-shading layer cannot prevent bad influences of inverse diffusion of the reagent into the specific binding layer and permeation of an enzyme reaction product formed in the specific binding layer into the detection layer. Each of DE 3227474 and EP 236768 discloses a specific binding assay device which comprises multilayer analytical materials into which the principle of the homogeneous method is introduced or in which an immobilized enzyme substrate is introduced into a specific binding layer. However, both of these devices cannot avoid the limitations of the homogeneous method described in the foregoing, and the bad effects caused by the curved binding distribution pattern of a label in the specific binding layer are very serious because of the short and thin chromatography layer.
As described above, a number of specific binding assay methods have been developed with the aim of improving simplicity and quickness, but virtually nothing has been reported about a highly sensitive specific binding assay method which can also be used for various purposes and which can determine a substance to be assayed semi-quantitatively or quantitatively with high accuracy.
With the recently expanding domestic and regional medical care and increasing demands for emergency clinical inspections, great concern has been directed toward the development of a specific binding assay method by which a substance to be assayed can be measured quickly, easily and accurately even by persons who have no knowledge about clinical inspection. Under such circumstances, various types of sensors have been developed including electrochemical sensors. In the field of biochemical assay methods, simple and quick measuring methods have been developed making use of enzyme sensors and the like, and have already been disclosed for example in JP-B 02-59424 (the term "JP-B" as used herein means an "examined Japanese patent publication") and EP 125136. Though positive attempts have been made to apply such measuring methods to the specific binding assay process, virtually nothing has been reported on the development of a general purpose assay process which can be handled easily. Of these prior art sensors for use in the specific binding assay, electrochemical sensors which are effected by the use of relatively high sensitivity labels such as enzymes are described in the following.
EP 125136 discloses a specific binding reaction on the surface of an electrode to which an enzyme (catalase)-labeled antibody preparation is applied. Also, EP 125136 discloses a competitive specific binding reaction on the surface of an electrode to which an enzyme (glucose oxidase)-labeled substance is applied. Since a specific binding substance is immobilized on the surface of an electrode which has a limitation in its size, each of the sensors in these disclosures is limited in the immobilization efficiency. Because of this, and because the reaction is effected in a liquid phase, each of these sensors possesses disadvantages in terms of sensitivity and measuring time.
EP 125136 discloses an enzyme activity detection sensor which comprises an oxidation-reduction enzyme (glucose oxidase or the like) and an electron mediator (a ferrocene derivative or the like), in which the extent of a specific binding reaction is measured using either of the enzyme and the electron mediator as a label, by changing (a) the effective level of the electron mediator, (b) the effective level of the enzyme, (c) the effective levels of the electron mediator and the enzyme simultaneously and (d) the effective area of the electrode. According to this disclosure, the specific binding reaction is effected on the surface of a solid phase (vessel wall or electrode) (heterogeneous method) or in a solution where the electrode exists (homogeneous method). Similar to the case of other heterogeneous type methods, the heterogeneous method of this disclosure requires a step for the isolation or washing of unreacted substances. In addition to this, only the surface of the vessel wall or the electrode is available as the immobilization carrier. This sensor, therefore, fails to improve the measuring efficiency and simplicity of the heterogeneous method. Also, the homogeneous method of this disclosure is substantially identical to any of the aforementioned homogeneous type methods, thus leaving the homogeneous method-inherent problems unsolved. For example, in the homogeneous method in which a ligand-modified electron mediator is used, the electron mediator should have a capacity to perform electron transfer by getting close to the redox center of the enzyme molecule. However, when an electron mediator is modified with a ligand, the function of the electron mediator is spoiled in some cases depending on the type of the ligand to be used, thus entailing limited availability of ligands, as well as a disadvantage in terms of the sensitivity. A similar technique has been disclosed in EP 402126 which also has a limitation with regard to the binding of an electron mediator and a ligand to carrier molecules.
Similar techniques have also been disclosed for instance in EP 142301, EP 125139, EP 150999, JP-A 63-139248 (the term "JP-A" as used herein means an "unexamined published Japanese patent application"), U.S. Pat. No. 4,963,245, U.S. Pat. No. 5,066,372, Anal. Chem. (vol. 56, pp. 2355-2360, 1984) and Clin. Chem. (vol. 31, pp. 1449-1452, 1985). Of these, each of U.S. Pat. No. 4,963,245 and U.S. Pat. No. 5,066,372 discloses a semi-homogeneous method in which the measurement after a specific binding reaction is effected without requiring a step for the washing of unreacted substances, by magnetically separating specific binding substance-immobilized magnetic particles on the surface of an electrode. However, this type of method requires a magnetic separation device in order to separate the magnetic particle carrier physically. In addition, similar to the case of the aforementioned proximal linkage immunoassay (enzyme channeling technique or the like), a labeled enzyme free in the liquid phase can approach the electrode by free diffusion, and the resulting non-specific reaction becomes a cause of problems.
EP 223541 discloses a heterogeneous electrochemical specific binding assay process in which an enzyme (alkaline phosphatase) is used as a label that catalyzes conversion of an inactive type electron mediator precursor (a ferrocene-linked substrate) into an active type electron mediator. A PCT application (WO 89-05454) discloses a heterogeneous type specific binding assay process in which an enzyme (alkaline phosphatase) that catalyzes conversion of a precursor (NADP.sup.+) into its oxidation-reduction pair (NAD.sup.+) is used as a label, and the amount of the formed oxidation-reduction pair is measured using an electrochemical enzyme sensor. Also, JP-A 02-112752 discloses a process in which biological cells trapped on a depth filter are allowed to react with enzyme-labeled antibody on the filter, unreacted molecules of the labeled antibody are removed by washing, and then the biological cells are detected by measuring activities of the labeled enzyme using an electrode. However, being a typical heterogeneous method, each of these processes fails to improve the measuring performance and simplicity of the conventional heterogeneous method.